Determining Dilution Accuracy in Microtiter Plate Assays

Using the Artel MVS® Multichannel Verification System

John Thomas Bradshaw, Ph.D.Senior Development Scientist

• Title: Determining Dilution Accuracy in Microtiter Plate Assays Using the Artel Multichannel Verification System (MVS®)

• Presenter: John Thomas Bradshaw, Ph.D.

• Description: Many critical assays are based upon conducting accurate dilutions of key reagents. Accurately knowing sample concentration is critical for properly interpreting experimental results, which can only be obtained if the experimental dilution ratio is known and controlled. Herein we present an application of the MVS which provides a NIST traceable, dual-dye absorbance method for determining the accuracy of each step in a dilution process. The measurements provided by this method allow for testing both single and multiple point dilutions, and cover a testable dilution range of up to 1/2000. Both theory and experimental validation of this method will be discussed.

Workshop Outline

• Artel

• Dual-Dye Photometry and the Artel MVS

• Dilution Application using the MVS

• Conclusions

• Leader: Liquid handling quality assurance

• Focus: Laboratory quality & productivity

• Core Technology: Ratiometric PhotometryTM

Ratiometric Photometry• Technology used for both the ARTEL PCS® Pipette

Calibration System and the ARTEL MVS®

Multichannel Verification System:

• Photometric measurement of liquid volume• Two specially-formulated dyes measured at two wavelengths • Ratiometric measurements and calculation of results• Simultaneous measurement of accuracy & precision per

channel

Mix

Photometric Measurements

Dispense Sample

Dispense Diluent

MVS® Multichannel Verification System

• Verifies liquid handlers from any manufacturer

• Standardized measurement platform

• Traceable results — repeatability and standardization

• Accuracy and precision per channel

MVS Components

Characterized Microtiter Plates

Sample + Diluent Solutions

Calibrator Plate

Plate Shaker Notebook Computer w/ System Software &

Barcode ReaderMicrotiter Plate Reader

MVS Performance

Characterized Microtiter

Plates

• Optimizes liquid handling instrumentation

• Complete, integrated, mobile system

Sample Solutions

Software

Calibrator Plate

MVS – Features

• Extremely accurate and precise

• Exceptional for low volumes – down to 0.03 µL

• Easy to use and convenient

• Fast – 5-10 minutes

• Traceable to national standards (NIST)

• ISO-approved method

• Compliance with 21 CFR

• Helps maintain experimental integrity and confidence

MVS is the Standard Platform

• Components of the Artel MVS work together to provide accuracy and precision assessment for target volumes dispensed into microtiter plates.

• Standardization between liquid handlers and/or laboratories really means…

2 µL dispensed by a Tecan Evo in

Harlow, UK on Tuesday

2 µL dispensed by a Beckman FX in

Collegeville, PA on Thursday

2 µL dispensed by a PerkinElmer JANUS in Research Triangle Park, NC on Friday

= =

MVS Summary

• The MVS ratiometric absorbance method provides an easy way to assess the volume delivered from a liquid handler

• Traceability of MVS results = standardization across multiple laboratories and equipment

• Fast & Easy• Accurate & Precise• Automation-ready

• and now, MVS measures dilution accuracy

Dilution Application using MVS

Vs1Vs2

V1

Vs2 + Vd2

Vs3

V2 Vs4

V3Vs3 + Vd3

Vs2

Vd2Vs3

Vd3Vs4

Well 1

Well 2

Well 3

Vs1

What are Dilutions?• Controlled reduction in concentration of an analyte

of interest• Critically important process in most laboratories• Accuracy is imperative at each step• A common dilution protocol involves multiple serial

dilution steps over a defined rangeInitial

SampleDilution Step 1

1:2

Dilution Step 21:20

Dilution Step 31:1000

1:2 1:10 1:50

Applications for Serial Dilutions• Primary screening/compound

management

• Secondary screening/ADME-Tox

• Dose response

• Viral loading

• HIV testing

• Other clinical applications

• Bacteria isolation

• And many others…

• The accuracy of many of these experiments is dependent upon knowing the final concentration of some analyte(s).

Dilution Testing with the MVS:Review of MVS Components

Sample Solutions

Diluent Solution

MVS Sample Solutions

• Contain 2 dyes, red and blue

• Distinct absorbance maxima (520 & 730 nm)

• Different concentrations of red dye for different volume ranges

• Blue dye at the same concentration for all ranges

• Stable and traceable to national standards

MVS Diluent

• Contains blue dye only

• Absorbance maximum at 730 nm

• Concentration of blue dye same as in sample solutions

• Used to fill wells to working volume

• Stable and traceable to national standards

Diluting MVS Sample Solutions with Diluent

+ =• Variable Red Dye

concentration

• Uniform Blue Dye concentration• any dilution made results

in no change to the blue dye concentration

MVS Dilution Foundation: Beer’s Law

• MVS Dilution calculation uses a foundation based on the Beer-Lambert Law:

• aλ is the absorbance per centimeter of a solution with known dye concentration

( ) lalCA ⋅== λλλ ε

Absorbance per Pathlength for MVS Dyes

• The absorbance per pathlength for Red dye, which is in Sample Solution only:

rr Ca ⋅= 520ε

• The absorbance per pathlength for Blue dye, which is the same for Sample and Diluent Solutions:

bb Ca ⋅= 730ε

Sample Solutions

Diluent Solution

Vs1Vs2

V1

Vs2 + Vd2

Vs3

V2 Vs4

V3Vs3 + Vd3

Vs2

Vd2Vs3

Vd3Vs4

Well 1

Well 2

Well 3

Vs1

MVS Dilution Theory(JALA, October 2007, vol 12, pp 260-266)

b

r

aaAA ⋅= 1,7301,520

⎟⎟⎠

⎞⎜⎜⎝

⎛+

⋅⋅=22

22,7302,520

ds

s

b

r

VVV

aa

AA

⎟⎟⎠

⎞⎜⎜⎝

⎛+

⋅⎟⎟⎠

⎞⎜⎜⎝

⎛+

⋅⋅=33

3

22

23,7303,520

ds

s

ds

s

b

r

VVV

VVV

aaAA

Accuracy of Dilution Steps

m

n

n

mmn A

AAA

R,730

,730

,520

,520 ⋅=

• When dilution steps result in measurable absorbance values for both red and blue dyes, then:

Initial Sample

Dilution Step 1

1:2

Dilution Step 2

1:4

Dilution Step 3

1:8

well m well n

Large Dilution Steps

n

n

b

rn A

AaaR

,520

,7301 ⋅=

• When large dilution steps are performed, highly concentrated red dyes are used.

• Plate reader cannot measure the absorbance in well 1.• Absorbance per pathlength values known for red & blue

dyes

Initial Sample

well 1 well n

Dilution Step 11:100

MVS Dilution MeasurementExample 1: An 11-Step Serial 1:2 Dilution

• Starting with neat solution in column 1, serial 1:2 dilutions will be made across a microtiter plate, to a final dilution of 1:2048

Conducting a 1:2 Dilution

• Column 2: 100 µL Sample + 100 µL Diluent = 200 µL Total

• Mix

• Aspirate 100 µL from the 200-µL mixture

• Dispense 100 µL into next column

• In column 2, red dye is now ½ as concentrated

• Repeat…in column 3, the dye is now ¼ as concentrated

• After multiple steps, the red dye becomes too dilute to measure………so the process is repeated with a different (more concentrated) starting red dye solution

1:400 – 1:2000740Range E1:100 – 1:400185Range D1:20 – 1:10075Range C1:4 – 1:2015Range B1 – 1:43.75Range A

Dilution RangeRed dye absorbance per pathlength (cm-1)

Sample Solution

Dilution Range for MVS Sample Solutions

( )Car ⋅= 520ε

• To cover all 11 steps of a 1:2 serial dilution protocol, all range solutions will be used

Parameters of 1:2 Serial Dilution Example

• 96-well plate

• 8-channel liquid handler, dispensing left to right

• 1:2 dilution across plate, 11 steps

• Starting with 200 µL in first column (with neat reagent, i.e., Range A, B, C…)

• Transfer volume = 100 µL

• Columns 2-12 contain 100 µL Diluent

Plate 1 (Range A): Set up

200 µL neat Range A solution

100 µL Diluent

Plate 1 (Range A): First Step100 µL transfer to

column 2; mix with pipette

100 µL remaining 200 µL Total

Plate 1 (Range A): Remaining Steps100 µL transfer to

column 3; mix with pipette

100 µL to waste

100 µL Total in every well

Dilution IDs for 11-step 1:2 Serial Dilution

1:1 1:161:2 1:81:4 1:32 1:64

1:128

1:256

1:512

1:1024

1:2048

Repeat Serial 1:2 Dilution Steps for Each Range Solution – 5 plates required

Range APlate (1)

Range BPlate (2)

Range CPlate (3)

Range DPlate (4)

Range EPlate (5)

Based on definition of plate layout and dilution values, MVS “grabs” necessary information values from each plate

Dilution IDs for Each Range Solution:5 plates required

1:1 1:161:2 1:81:4 1:32 1:64

1:128

1:256

1:512

1:1024

1:2048

Range Aplate

Range Bplate

Range Cplate

Range Dplate

Range Eplate

Other Step Dilution Examples• 1:2 –

• 150 uL into 150 uL (300 uL total), remove 150 uL

• 50 uL into 50 uL (100 uL total), remove 50 uL

• 1:3 –

• 50 uL into 100 uL (150 uL total), remove 50 uL

• 100 uL into 200 uL (300 uL total), remove 100 uL

• 1:5 –

• 20 uL into 80 uL (100 uL total), remove 20 uL

• 40 uL into 160 uL (200 uL total), remove 40 uL

• 1:10 –

• 10 uL into 90 uL (100 uL total), remove 10 uL

• 30 uL into 270 uL (300 uL total), remove 30 uL

Critical Influences on Dilution Accuracy

• Accurate transfer of volume at each step (!!!!)

• Mixing (!!!!!)

• Before transferring, if each well is not optimally mixed, errors will propagate with each successive step, compounding the error.

• Proper methodology or system to measure dilution steps

Dilution Guide: MVS Data Manager Software

• MVS Data Manager software controls the volume verification and dilution testing processes

• Immediate output reports with pass/fail results• Manual or auto-exporting (HTML, XML) • Data bases can be networked• 21 CFR Part 11 compliance ready• Automation compatible for in-process volume checks

Defining the Liquid Handler

• 8 or 12 channels• Use general or specific descriptors

Defining the Liquid Handler

• Liquid handler IDs are stored for repetitive testing

Plate Layouts Define Dilution Protocol

• Define the test parameters (plate format, target dilutions, no. of channels & reps, tolerances)

• Each dilution value may have different tolerance specifications for accuracy and precision

• Plate layout templates are stored for repetitive testing

MVS Software Guides User During Verification

Select plate layout and plate type

Select device to testScan all required materials

Dispense reagents w/ liquid handler

Shake plate for uniform meniscusPlate Reader measurements

After Measuring 5 plates…MVS Output Report (1 of 3)

Data Manager Output Report

(2 of 3)

Data Manager Output Report

(3 of 3)

Conclusion

• MVS aides in optimizing performance of automated liquid delivery systems

• Fast, easy, accurate and precise methodology for productivity, repeatability and data integrity

• Standardized system and reagents allows for dilution testing up to a dilution of 1:2000

• The new MVS serial dilution capability supports critical liquid handling processes in life science laboratories

Please visit us in Booth #551 for a demonstration of our products.

Thank You